DNA replication is precisely regulated to achieve duplication of the genome exactly once before each cell division. Considerable progress has been made in the identification of the proteins involved in regulating replication, which are highly conserved from yeast to humans. In yeast, pre-replication complex (pre-RC) proteins, which include the origin recognition complex (ORC) and minichromosome maintenance (Mcm) proteins, assemble at specific DNA sequences that function as replication origins. In mammals, specific ORC binding sites have not been identified, nor has any specific sequence of bases been shown to direct initiation of DNA replication. Nonetheless, replication initiates at specific and reproducible locations within mammalian chromosomes. We are interested in the means by which mammalian cells select these origin sites. We have exploited a cell-free replication system in which Chinese hamster ovary (CHO) cell nuclei staged at various times during G 1-phase are stimulated to initiate replication by introduction into extracts from Xenopus eggs. Using this system, we have identified a distinct point during Gi-phase (Origin Decision Point; ODP) when specific origin sites are established from amongst a larger set of potential sites. The ODP takes place after the assembly of pre-RCs but prior to the restriction point and the activation of S-phase promoting kinases. Our current working hypothesis is that pre-RCs form at many sites that initially have an equal potential to serve as replication origins. Subsequent events potentiate some sites and/or inactivate others. In this application, we propose to test two important predictions of this model. First, our studies of origin specification have so far been limited to the dihydrofolate reductase (DHFR) gene locus but we predict that a similar selection process takes place at most or all loci. Here, we will determine whether other replication origins also are specified at a particular time during GI-phase and whether there is a single ODP for all origins or whether different origins are specified at different times during Gi-phase. Second, we predict that there should be more sites of pre-RC formation than active replication origins. Using a combination of chromatin immunoprecipitation and nascent DNA strand labeling methods, we will identify the chromatin binding sites for pre-RC proteins within the DHFR locus throughout the cell-cycle and relate this to the sites of initiation of DNA replication during S-phase.